STRUCTURE OF THE DISTAL HUMAN GONADOTROPIN-RELEASING-HORMONE (HGNRH) GENE PROMOTER AND FUNCTIONAL-ANALYSIS IN GT1-7 NEURONAL CELLS

To assess potential species-specific expression of gonadotropin releas ing hormone (GnRH), the distal human (h) GnRH promoter was cloned, cha racterized and tested in gene transfer studies. The nucleotide sequenc e of similar to 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter reg ion between -551 h (-424 r) and the transcriptional start site and wit hin multiple distal promoter regions. In contrast, there was little si milarity in the sequences between -1131/-551 h and -1031/-424 r. A del etion panel of 5'-flanking hGnRH promoter constructs was made and test ed in transient transfection assays in GnRH-producing mouse GT1-7 neur onal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibite d high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the ra t gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase repor ter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assay s showed unique protection patterns with nuclear extracts from GT1-7 a nd Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. The se data suggest the presence of different cis-acting elements and tran sacting factors that mediate species-specific neuronal GnRH expression .